雌二醇通过雌激素受体α介导调控Cyclin D1在小鼠前列腺平滑肌细胞中的表达

目的 观察雌激素受体α(ERα)在17β-雌二醇(E2)促进小鼠前列腺平滑肌细胞(PSMC)增殖中的作用,探讨E2对PSMC的作用机制.方法 构建pGSadeno-ERα腺病毒载体,体外转染原代培养的小鼠前列腺平滑肌细胞(PSMC),将实验细胞分组:实验组;ERα基因下调组,细胞转染ERα干扰序列腺病毒;阴性对照组,细胞转染无义序列腺病毒;正常对照组,正常的小鼠PSMC.各组分别加入1×10-8mol/L E2,72 h后流式细胞仪检测细胞增殖,Western blot检测细胞内Cyclin D1基因表达.结果 E2作用细胞72 h后,实验组细胞G0/G1期的比率(71.76±1.78)%显著高于阴性对照组(51.73±1.86)%和正常对照组(47.90±0.79)%,差异有统计学意义(P＜0.05);相对应的PI值:实验组(28.24±1.78)显著低于阴性对照组(47.27±1.86)和正常对照组(52.10±0.79)比较,差异有统计学意义(P＜0.05);实验组细胞内Cyclin D1基因表达水平显著低于对照组.结论 雌激素在对前列腺平滑肌细胞的生物学效应中,通过雌激素受体α的介导调控了细胞周期素Cyclin D1的表达,从而促进细胞的增殖.

Abstract：

Objective To investigate the role of estrogen receptor alpha (Erα) in estradiol-induced proliferation of mouse prostatic smooth muscle cells (PSMCs) and explore a new target for benign prostazic hyperplasia (BPH). Methods Adenoviral vectors with Erα-shRNA or Ctrl-shRNA were transfected into mouse PSMCs. The cells were divided into 3 groups: experimental group ( cells transfected with pGSadeno-Erα), negative control group (cells transfected with pGSadeno-Ctrl) and normal control group (normal mouse PSMCs). The expression of Cyclin DI was detecrted by Western blotting. Cell cycle was analyzed by using flow cytometry. Results After treatment in the presence of 1 10-8 mol/L 17β-estradiol (E2) for 72 h, the percentage of cells in the G0/G1 phase was (71.76 1.78)％ in experimental group, which was significantly higher than in negative control group (51.73 ± 1.86)％ and normal control group (47.90±0.79)％, and PI in experimental group (28.24±1.78) was prominently lower than in negative control group (47.27±1.86) and normal control group (52.10±0.79). The expression level of Cyclin D1 in experimental group was conspicuously lower than in control groups. Conclusion E2 regulates the expression of Cyclin D1 via Erα in PSMCs, which promotes cell proliferation.     

人胰岛素样生长因子1基因在海绵体平滑肌细胞中的表达及定位

目的观察人胰岛素样生长因子1基因在海绵体平滑肌细胞中的表达及定位情况,为阴茎勃起功能障碍的基因治疗提供理论和实验依据。方法采用Lipofectamine2000包裹PCDB-hIGF-1质粒和PCDB空载体,转染原代培养的大鼠阴茎海绵体平滑肌细胞,转染后24h分别采用RT-PCR法检测hIGF-1基因mRNA表达,Westernblot法检测hIGF-1蛋白表达,荧光免疫细胞化学法检测hIGF-1细胞定位及蛋白表达。结果RT-PCR检测到hIGF-1mRNA表达,Westernblot和荧光免疫细胞化学法均检测到hIGF-1蛋白表达,荧光免疫细胞化学法可见hIGF-1蛋白定位于阴茎海绵体细胞胞质中。结论PCDB-hIGF-1成功转染入大鼠阴茎海绵体平滑肌细胞,大鼠阴茎海绵体平滑肌细胞可表达hIGF-1基因,蛋白定位于胞质。     

RNA干涉胰岛素样生长因子1类受体的研究

RNAi技术的关键是siRNA的制备，实验结果证实，通过发卡结构的小RNA（shRNA）载体表达siRNA可以抑制特定靶基因表达。我们通过将针对IGFIR基因的RNA干扰片段装入特定质粒PSUPER中，再转染入人肝癌细胞株SMMC7721中，并筛选出稳定株。     

转染胰岛素样生长因子-1基因对原代培养的阴茎海绵体平滑肌细胞增殖的影响

目的 观察转染胰岛素样生长因子-1( IGF-1)基因对原代培养的阴茎海绵体平滑肌细胞(CCSMCs)增殖的影响,探讨IGF-1基因治疗勃起功能障碍(ED)的可能机制.方法 构建pcDNA3.1-IGF-1,体外原代培养大鼠CCSMCs并免疫组织化学染色鉴定;CCSMCs分3组:转染pcDNA3.1-IGF-1组、pcDNA3.1组和对照组,转染后不同时间段(3、5、7、9d)噻唑蓝(MTT)比色法检测细胞增殖,同时不同时间段( 24、48、96 h)酶免疫分析法(EIA)定量检测CCSMCs分泌IGF-1含量的变化.结果 成功克隆pcDNA3.1-IGF-1载体,成功原代培养CCSMCs;转染pcDNA3.1-IGF-1组细胞在转染后5、7、9d(分别为0.72±0.08、0.80±0.06、0.82 ±0.07)较转染pcDNA3.1组(分别为0.58±0.07、0.63±0.05、0.61 ±0.08)和对照组(0.59 ±0.06、0.61 ±0.07、0.62 ±0.03)增殖明显增加,呈时间依赖性,差异有统计学意义(P＜0.05).与转染pcDNA3.1组[分别为(46±7)、(48±9)、(50±11) μg/L]和对照组[分别为(47±11)、(52±10)、(51±10) μg/L]比较,转染pcDNA3.1-IGF-1组细胞在转染后24、48、96 h CCSMCs分泌的IGF-1含量[分别为(58±8)、(69±11)、(71 ±8) μg/L]增加,差异有统计学意义(P＜0.05).结论 转染IGF-1基因可提高CCSMCs的增殖能力和增加IGF-1的分泌,可能是IGF-1基因治疗ED的基础.     

siRNA封闭胰岛素样生长因子Ⅰ类受体对肝癌细胞侵袭能力的影响及其机制

目的 探讨人胰岛素样生长因子1类受体(IGFIR)的短发夹环RNA质粒(pSUPER-siRNA-IGFIR)能否有效抑制人肝癌细胞株MHCC97H侵袭能力并探讨其相关机制.方法 设计并合成靶向IGFIR的siRNA片段并构建Psuper-siRNA-IGFIR表达质粒,将其转入MHCC97H、SMMC7721细胞,通过G418筛选出稳定株.通过黏附实验、Boyden小室实验、软琼脂克隆形成实验观察各细胞株侵袭能力的变化并采用明胶酶法测定肝癌细胞的金属蛋白酶(MMP)-2、MMP-9的含量,Western blot检测增殖相关基因的变化.并设空白对照组、阳性对照组.结果 MHCC97H-IG-FIR-siRNA、SMMC7721-IGFIR-siRNA黏附能力比对照组下降约5倍(MHCC97H)和6倍左右(SMMC7721),细胞迁移能力明显下降,下降各约3倍,细胞克隆形成率明显低于对照组约6倍和8倍,其MMP-2、MMP-9、ICAM-1、LNR、E-cadherin表达比对照组低.结论 构建的pSUPER-siRNA-IGFIR具有RNA干扰作用,可抑制肝癌细胞株转移能力.     

雌激素对大鼠前列腺平滑肌细胞增殖的影响

目的 观察17β-雌二醇(E2)对大鼠前列腺平滑肌细胞(PSMC)增殖的影响.方法 取体重(253±28)g的雄性SD大鼠30只,无菌切取前列腺,应用酶消化法行原代细胞培养.取3～4代传代细胞,分别加入不同浓度E2(0.1～100)nmol/L处理72 h,应用流式细胞仪检测细胞周期、细胞凋亡及其相关蛋白Cyclin D1,应用western blot法检测bcl-2和bax表达.结果 E2(1、10 nmol/L)促进PSMC从G1期向S期过渡,其S期细胞比率分别为(18.50±4.98)%、(21.16±4.83)%,显著高于对照组(12.39±2.64)%(P＜0.05),并伴随Cyclin D1蛋白表达显著增高.而高浓度E2(100 nmol/L)则抑制细胞增殖,其S期细胞比率为(7.98±1.92)%,显著低于对照组(P＜0.05),并伴随bax表达显著增加和细胞凋亡率显著升高.结论 低浓度E2能够上调CyclinD1表达加速G1期向S过渡从而促进大鼠PSMC增殖,高浓度E2则通过增加bax表达促进细胞凋亡.     

胰岛素样生长因子-1及其受体在前列腺癌组织的表达及其临床意义

目的:探讨胰岛素样生长因子-1(IGF-1)及其受体(IGF-1R)在前列腺癌(PCa)组织中的表达及其临床意义。方法:取2017年1月至2019年12月河北医科大学第三医院泌尿外科的41例前列腺癌患者的前列腺组织作为实验组;并收集21例良性前列腺增生(BPH)患者前列腺组织当做对照组;并通过前列腺特异性抗原(PSA)...     

胰岛素样生长因子—ImRNA在前列腺组织中的表达

对１５例良性前列腺增生和１２例正常前列腺组织标本中胰岛素样生长因了－Ｉ（ＩＧＦ－Ｉ）ｍＲＮＡ的表达情况进行了研究。ＲＴ－ＰＣＲ的方法可自所有标本中检出ＩＧＦ－Ｉ ｍＲＮＡ的表达，并用电泳和Ｓｏｕｔｈｍ杂交证实了结果的可靠性。     

前列腺癌患者血清胰岛素样生长因子-1检测的临床意义

目的　探讨血清胰岛素样生长因子 1(IGF Ⅰ )与前列腺癌 (PCa)发生发展的关系。　方法　采用免疫放射分析法 (IRMA)检测 3 7例PCa、3 5例良性前列腺增生 (BPH)患者和 2 0例健康人血清IGF Ⅰ ,比较各期PCa血清IGF Ⅰ水平 ,并对 8例行根治性前列腺全切术后患者手术前后IGF Ⅰ水平随访。　结果　PCa组血清IGF Ⅰ ( 3 2 5 .6± 10 0 .8)ng/ml,明显高于BPH组 ( 2 0 1.6± 5 3 .8)ng/ml和健康组 ( 179.0± 5 7.2 )ng/ml,差异有显著性意义 (P <0 .0 1) ;BPH组与健康组比较差异无显著性意义 (P >0 .0 5 ) ;8例PCa患者术前IGF I( 3 15 .8± 87.0 )ng/ml,术后 ( 2 2 4.8± 88.4)ng/ml,差异有显著性意义 (P <0 .0 5 ) ;PCa患者各期血清IGF Ⅰ比较差异无显著性意义 (P >0 .0 5 )。　结论　IGF Ⅰ有可能作为临床上一个新的PCa检测指标预测高危人群 ,进行早期诊断     

原位杂交检测胰岛素样生长因子Ⅰ和Ⅱ在前列腺组织中的表达

为了解胰岛素样生长因子Ⅰ和Ⅱ在正常和增生前列腺组织中的表达情况，作者利用分子原位杂交的方法对正常和良性增生的前列腺组织中ＩＧＦ－Ｉ和ＩＧＦ－Ⅱ的ｍＲＮＡ的表达情况进行了研究。表明，正常和增生的前列腺组织中均有ＩＧＦ－ＩＧＦ－ⅡｍＲＮＡ的表达。     

Human prostatic smooth muscle cells in culture: Estradiol enhances expression of smooth muscle cell-specific markers

Smooth muscle cells (SMCs) constitute a major cellular component of prostatic stroma. SMC tension plays an important role in urethral obstruction secondary to benign prostatic hyperplasia (BPH). We have developed an in vitro procedure for the propagation of human prostatic SMCs. Tissue specimens from patients undergoing radical prostatectomy or cystectomy were enzymatically disaggregated and cultured in MCDB-131 medium supplemented with horse serum, insulin, conditioned medium from the tumor cell line CRL-5813, and steroid hormones. The medium was assembled on the basis of the effects these supplements have on the growth of SMC cultures and on the expression of the two markers desmin and smooth muscle myosin. Addition of 0.1 μM of estradiol to the growth medium dramatically increased expression of these SMC-specific markers. Dihydrotestosterone (DHT) and hydrocortisone had a similar, albeit less pronounced effect. At three to five passages, about two thirds of the cells were immunohistologically positive for smooth muscle myosin or desmin. Almost all cells were positive for the myofibroblast marker smooth muscle α-actin throughout 10 passages and more. In SMC cultures, cells staining for smooth muscle myosin and desmin were found to seek direct contact to myofibroblasts. They grew in aggregates on a layer of myofibroblasts which adhered to the surface of the culture vessel. As revealed by transmission electron microscopy the cultured cells exhibited morphological features of myofibroblasts. Characteristics of smooth muscle cells, such as prominent bundles of microfilaments associated with dense bodies, basal laminae investing the cells, and numerous caveolae at the cell surfaces were regularly observed in cultures of low passages. After several passages, these features were markedly decreased and organelles of the biosynthetic system became more prominent. In summary, we present an in vitro model of prostatic SMCs and demonstrate that steroid hormones have characteristic effects on these cells. SMC cultures are expected to facilitate investigation of the functions and properties of human prostatic SMCs. Prostate 30:117–129, 1997. © 1997 Wiley-Liss, Inc.     

Regulation of urogenital smooth muscle patterning by testosterone and estrogen during prostatic induction

BACKGROUND: Smooth muscle (SM) has been proposed to play an important role in controlling prostate organogenesis by regulating signaling between inductive mesenchyme and developing epithelial prostatic buds. METHODS: We have examined the effects of testosterone and estrogen upon SM patterning in the embryonic rat urogenital tract (UGT) using in vitro organ cultures, immunohistochemistry, and Western blotting. RESULTS: We observed that testosterone elicited a sexually dimorphic difference in SM structure of embryonic UGTs, in cultures grown with testosterone. The addition of estrogen led to an increase in the rate of SM closure, in both males and females. To quantify the effects of steroids upon SM we used Western blotting of SM actin, which showed that estrogen stimulated SM content, while testosterone reduced SM content. Finally, we examined the expression of ERalpha, ERbeta, PR, and SM actin under different hormonal treatments of UGTs grown in vitro. The expression patterns of ERalpha and ERbeta were largely unchanged by hormonal treatment, while PR showed a much broader expression pattern in response to estradiol. CONCLUSIONS: Our results indicate that testosterone can directly regulate SM patterning and content in the UGT, and that SM is sensitive to both androgens and estrogens.     

The alpha-adrenoceptor subtype mediating the tension of human prostatic smooth muscle

We have characterized the α₁ adrenoceptor subtypes in the human prostate using radioligand receptor binding studies. The objective of the present study was to determine the α₁ subtype mediating the tension of prostatic smooth muscle. Fresh human tissue was obtained from 9 males between 50 and 80 years of age undergoing prostatectomy for BPH. The incubation of prostatic tissue with the irreversible antagonist chlorethyclonidine (CEC) resulted in an 80% reduction of the maximal contractile response produced by phenylephrine. However, the α_1A-selective antagonists WB-4101 and 5-methylurapidil (5-MU) competitively inhibited the contractile response induced by phenylephrine, with K_B = 2.64 and 4.46 nM, respectively, consistent with their affinity at the α_A1 receptor subtype. The pharmacological profile of the α₁-receptor-mediated contractile response of prostate smooth muscle is inconsistent with their classification as either an α_1A or α_1B subtype. Alternatively, when compared with the properties of the cloned α₁ receptors, our results suggest that the α₁ receptors involved in the contraction of prostate smooth muscle have some pharmacological properties similar to those encoded by the gene of the bovine α_1c receptor subtype. The findings of the present study suggest that efforts should be made to confirm the identity of the α₁-receptor subtype expressed by prostate smooth muscle, in order to develop subtype-selective α₁ antagonists, and to evaluate their safety and efficacy in benign prostatic hyperplasia (BPH). © 1993 Wiiey-Liss, inc.     

Nicotine inhibits apoptosis and stimulates proliferation in aortic smooth muscle cells through a functional nicotinic acetylcholine receptor

Atherosclerosis and neointimal hyperplasia formation are induced by alterations in the homeostatic balance between cell growth and cell death. Apoptosis is a physiological cell death process that, when deregulated, may be involved in many pathological conditions. Cigarette smoking is a primary risk factor for vascular disease and nicotine seems to exert its atherogenic effects in part through the increase of smooth muscle cell (SMC) proliferation. The aim of this study was to investigate the effect of nicotine on SMC apoptosis. Nicotine added for 24 and 72 h to serum deprived cell cultures resulted in a decrease of apoptotic SMCs. The inhibition was direct and not mediated by platelet-derived growth factor, basic fibroblast growth factor, and transforming growth factor beta(1), autocrinally released by nicotine-treated SMCs, because it was not influenced by addition of specific neutralizing antibodies. Apoptosis inhibition as well as the proliferation increase, and basic fibroblast growth factor expression on nicotine-treated SMCs were blocked by nicotinic acetylcholine receptor antagonists, including alpha-bungarotoxin, a competitive antagonist of alpha subunits of nicotinic receptor. In conclusion, we propose that nicotine could lead to the increase of neointimal SMCs in vascular lesions by inducing the inhibition of physiological SMC apoptosis and the increase of SMC proliferation. We also showed that nicotine signaling occurs as a result of activation of the classical nicotine receptor pathways.     

Protease-mediated human smooth muscle cell proliferation by urokinase requires epidermal growth factor receptor transactivation by triple membrane signaling

Background

Urokinase (uPA) modulates cellular and extracellular matrix responses within the microenvironment of the vessel wall and has been shown to activate the epidermal growth factor receptor (EGFR). This study examines the role of the protease domain of uPA during EGFR activation in human vascular smooth muscle cells (VSMC).

Methods

Human coronary VSMC were cultured in vitro. Assays of cell proliferation and EGFR phosphorylation were examined in response to the carboxyterminal fragment of uPA (CTF) in the presence and absence of the plasmin, metalloprotease and a disintegrin and metalloproteinase (ADAM) inhibitors, heparin-bound epidermal growth factor (HB-EGF), and EGFR inhibitors, and small interfering RNA to EGFR and ADAMs.

Results

CTF produced a dose-dependent increase in DNA synthesis and cell proliferation in human VSMC, which was blocked in a dose-dependent manner by both plasmin inhibitors and the EGFR inhibitor, AG1478. CTF induced time-dependent EGFR phosphorylation, which was blocked by inhibitors of plasmin and metalloproteinases activity. The presence of urokinase plasminogen activator receptor was not required. Inhibition of ADAM-10 and -12, and of HB-EGF blocked EGFR activation in response to CTF. CTF-mediated activation of EGFR was mediated through Gβγ, src, and NAD(P)H oxidase.

Conclusions

In human coronary VSMC, uPA induces uPAR-independent, domain-dependent smooth muscle cell proliferation through transactivation of EGFR by a plasmin-mediated, ADAM-induced, and HB-EGF–dependent process, which is mediated by the intracellular pathways involving Gαi, Gβγ, src, and NAD(P)H oxidase.     

Identification of muscarinic receptor subtypes of cultured smooth muscle cells and tissue of human bladder body

BACKGROUND: Muscarinic receptor subtypes of cultured smooth muscle cells from the human bladder body were investigated by the receptor binding assay method. The result was compared with that obtained from the human bladder body tissue to confirm whether the receptor subtypes of the cells are not changed after several passages of cell culture. METHODS: Inhibitory effects of various muscarinic antagonists on the binding of [3H]-N-methylscopolamine ([3H]-NMS) to membrane preparations obtained from cultured smooth muscle cells from the fourth subculture of the human bladder body were compared with those prepared from the human bladder body tissue and cells expressing human muscarinic receptor subtypes. RESULTS: Binding-inhibition constants (pKi) for atropine, pirenzepine, methoctramine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), oxybutynin and propiverine obtained from membrane preparations of cultured smooth muscle cells were 8.91, 6.35, 8.24, 8.53, 7.29 and 5.61, respectively. pKi values of these muscarinic receptor antagonists against the membrane preparation of human bladder body tissue were 9.08, 6.66, 8.05, 8.79, 7.53 and 6.04, respectively. pKi values of cultured smooth muscle cells and tissue from human bladder body were correlated closely with those of insect cells expressing the cloned human M2 receptor subtype. CONCLUSION: The binding affinities for various muscarinic receptor antagonists of cultured human smooth muscle cells were maintained through the fourth subculture and it was suggested that the M2 receptor subtype is predominantly expressed in cultured smooth muscle cells of human bladder body as well as in tissue of the human bladder body.     

人脂肪干细胞向血管平滑肌细胞诱导分化的实验研究

目的：体外诱导培养人脂肪干细胞（adi pose-der i ved st em cel l s,ADSCs）,观察是否可以分化形成血管平滑肌细胞（vascul ar smoot h muscl e cel l s,VSMCs）,为构建组织工程化血管寻找新的种子细胞来源。方法：免疫磁珠法分选收集原代脂肪干细胞,加入PDGF-BB、TGF-β1诱导14天后进行检测。结果：实验组细胞生长呈现血管平滑肌细胞所特有的峰谷样生长,血管平滑肌细胞特有的表面抗原标记物表达阳性。结论：脂肪干细胞定向诱导培养后,具有血管平滑肌细胞的特性,有可能成为构建组织工程化血管新的种子细胞来源。